The lager beer fermentation consists of two fermentation steps: the primary fermentation for production of ethanol and flavor compounds, and the second fermentation (or lagering period) for maturation of the beer flavors. During the first fermentation, yeast cells produce α-aceto-α-hydroxybutyrate and α-acetolactate, which are intermediates for synthesis of isoleucine and valine, respectively. Some part of these acetohydroxy acids diffuses out of the cells, and turns into two vicinal diketones (VDK), 2,3-pentandione and diacetyl, respectively, by a non-enzymatic oxidative decarboxylation in the beer (see FIG. 1). VDK gives a butter-like unpleasant flavor, and the threshold for 2,3-pentandione and diacetyl are 0.9 and 0.15 mg/L, respectively (Meilgaard 1975; the details of the cited references will be described at the end of the specification).
Spontaneous decarboxylation of α-acetolactate is the rate-limiting step of diacetyl formation. The amount of VDK is reduced to acceptable levels during the lagering period. A reduction of the time needed for beer maturation may be achieved by use of a brewing yeast strain that produces less VDKs. Acetohydroxyacid synthase (Ilv2p/Ilv6p), acetohydroxyacid reductoisomerase (Ilv5p), dihydroxy-acid dehydratase (Ilv3p), and branched-chain amino acid aminotransferase (Bat1p)/transaminase (Bat2p) catalyze two homologous reactions for synthesis of isoleucine and valine.
There are two distinct approaches for the construction of yeast strains with lower VDK productions: an inactivation of acetohydroxyacid synthase and an enhancement of the acetohydroxyacid reductoisomerase activity. Down regulation with anti-sense RNA of ILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase decreases the enzymatic activity by more than 80%, and consequently reduces the diacetyl level at the fermentation mid-point by 40% (Vakeria et al. 1991).
On the other hand, the number of ILV5 gene encoding acetohydroxyacid reductoisomerase largely influences the VDK production. Yeast cells transformed with multicopy vectors carrying ILV5 show a five to ten-fold increase in the reductoisomerase activity and a concomitant decrease of diacetyl production by 50-60% compared to the control strain (Dillemans et al. 1987; Gjermansen et al. 1988). It is known that the enzymes involved in the synthesis of isoleucine and valine are located in the mitochondrial matrix (Ryan and Kohlhaw 1974). The mitochondria consist of two aqueous chambers (intermembrane and matrix) and two membranes (outer and inner membranes) (Wiedemann et al. 2003; Koehler 2004).
Since the mitochondria synthesize only eight stable proteins encoded by the mitochondrial DNA, most of the up to 1,000 mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytoplasm on free ribosomes as precursor proteins, and then transported into or across mitochondrial membranes with the aid of protein assembries known as TOM and TIM (translocases of outer and inner mitochondrial membranes, respectively (Endo et al. 2003; Truscot et al. 2003). The Ilv2p and Ilv5p are synthesized as precursor proteins in the cytoplasm, and then are imported into the mitochondrial matrix via TOM and TIM23 translocases.
The N-terminal presequence is cleaved off by a mitochondrial specific processing peptidase during translocation to the mitochondrial matrix (Gakh et al. 2002). The N-terminal 47 residues have been identified as Ilv5p cleavable presequence (Kassow 1992). Kassow describes that an ILV5 gene deleted in the region encoding the transit peptide was constructed, and the construct seems to have an effect on the diacetyl production level.
As commonly observed in typical mitochondrial targeting presequences, Ilv5p presequence is rich in positive charge, and its cleavage site is preceded by an arginine at the position −2 (von Heijne et al. 1989; Gakh et al. 2002). Ilv5p is known as a bifunctional protein that acts in mitochondrial DNA stability as well as in the biosynthesis of branched-chain amino acids (Zelenaya-Troitskaya et al. 1995). Mitochondrial DNA is inherited as a protein-DNA complex (the nucleoid). Together with other factors, Ilv5p maintains a stoichiometry between the mitochondrial DNA and the nucleoids by parsing the mitochondrial DNA into individual nucleoid in response to amino acid starvation (MacAlpine et al. 2000). The enzymatic function in branched-chain amino acid synthesis and the function for the mitochondrial DNA stability of Ilv5p can be separated by mutational approaches (Bateman et al. 2002). The enzymatic mutations map to conserved internal domains that are important for substrate and cofactor binding, whereas the mutations concerning the function for mitochondrial DNA stability map to the C-terminal region on the surface of Ilv5p. It is known that respiration-deficient (rho−) yeast produces elevated levels of VDK (Ernandes et al. 1993). This leads to a hypothesis that this is because acetohydroxyacid synthase (Ilv2p) does not properly localize in the mitochondria when it is “sick” (rho−), but rather tends to localize in the cytoplasm, where α-acetolactate is easily formed from cytoplasmic pyruvate.
Based on this idea, expression of a cytoplasmic Ilv5p has been explored in order to metabolize the cytoplasmically formed α-acetolactate (Kassow 1992). The cytoplasmic Ilv5p without N-terminal 47 residues complements auxotrophy of isoleucine but not of valine in an Ilv5Δ strain, indicating that a small portion of the truncated Ilv5p still localizes in the mitochondria supporting isoleucine synthesis.